Bottom Line:
The bioreactor was operated through two phases each of growth and expression.Cells successfully regrew during a 5-day growth phase.A combination of activity assays and Western blot analysis indicated production of an active and fully assembled tetramer of BChE.

ABSTRACTAn active and tetrameric form of recombinant butyrylcholinesterase (BChE), a large and complex human enzyme, was produced via semicontinuous operation in a transgenic rice cell suspension culture. After transformation of rice callus and screening of transformants, the cultures were scaled up from culture flask to a lab scale bioreactor. The bioreactor was operated through two phases each of growth and expression. The cells were able to produce BChE during both expression phases, with a maximum yield of 1.6 mg BChE/L of culture during the second expression phase. Cells successfully regrew during a 5-day growth phase. A combination of activity assays and Western blot analysis indicated production of an active and fully assembled tetramer of BChE.

Mentions:
Figure 4A shows a Western blot under reducing conditions of the cell-associated BChE from samples obtained during Expression 1 and Growth 2. Equal volumes (20 muL) of crude cell extract were loaded into each lane. These extracts were obtained by grinding cells in a 1:1 ratio of biomass to buffer, so the intensity of the BChE band corresponds to the concentration of BChE associated with the cells at the time each sample was taken. Under reducing conditions, a distinct band is seen around 85 kDa, which corresponds to the predicted size of a monomer of BChE. Samples taken immediately before and after induction and 2 days after induction (lanes 1–3) show no visible band, which corresponds to the low BChE activity as seen in the activity data shown in Figure 3D (< 5 mug/g FW). The band in lane 4 corresponds to a higher value of BChE activity (21 mug/g FW). However, while we see a decrease in BChE activity from day 20 to 21 (a drop from 21 to 18 mug/g FW), there is an increase in the intensity of the BChE band seen in the Western blot in lanes 5 and 6 (which correspond to samples taken immediately before and after initiation of Growth 2 on day 21). This may indicate that a portion of the BChE detected in the Western blot is inactive.

Mentions:
Figure 4A shows a Western blot under reducing conditions of the cell-associated BChE from samples obtained during Expression 1 and Growth 2. Equal volumes (20 muL) of crude cell extract were loaded into each lane. These extracts were obtained by grinding cells in a 1:1 ratio of biomass to buffer, so the intensity of the BChE band corresponds to the concentration of BChE associated with the cells at the time each sample was taken. Under reducing conditions, a distinct band is seen around 85 kDa, which corresponds to the predicted size of a monomer of BChE. Samples taken immediately before and after induction and 2 days after induction (lanes 1–3) show no visible band, which corresponds to the low BChE activity as seen in the activity data shown in Figure 3D (< 5 mug/g FW). The band in lane 4 corresponds to a higher value of BChE activity (21 mug/g FW). However, while we see a decrease in BChE activity from day 20 to 21 (a drop from 21 to 18 mug/g FW), there is an increase in the intensity of the BChE band seen in the Western blot in lanes 5 and 6 (which correspond to samples taken immediately before and after initiation of Growth 2 on day 21). This may indicate that a portion of the BChE detected in the Western blot is inactive.

Bottom Line:
The bioreactor was operated through two phases each of growth and expression.Cells successfully regrew during a 5-day growth phase.A combination of activity assays and Western blot analysis indicated production of an active and fully assembled tetramer of BChE.

ABSTRACTAn active and tetrameric form of recombinant butyrylcholinesterase (BChE), a large and complex human enzyme, was produced via semicontinuous operation in a transgenic rice cell suspension culture. After transformation of rice callus and screening of transformants, the cultures were scaled up from culture flask to a lab scale bioreactor. The bioreactor was operated through two phases each of growth and expression. The cells were able to produce BChE during both expression phases, with a maximum yield of 1.6 mg BChE/L of culture during the second expression phase. Cells successfully regrew during a 5-day growth phase. A combination of activity assays and Western blot analysis indicated production of an active and fully assembled tetramer of BChE.